The long-term goal of this project is an understanding of the costimulatory signals that antigen-presenting cells (APC) provide to naive T cells to regulate T cell antigen receptor (TCR)-driven clonal expansion and memory cell formation. CD28 and CTLA-4, which prefer CD86 and CD80 as ligands, are the most potent positive and negative costimulatory receptors for naive T cells, respectively. Blockade of CD28 is now approved for the treatment of rheumatoid arthritis, and inhibition of CTLA-4 is being tested in humans to enhance tumor immunity. Despite these promising advances, many questions remain as to how CD28 and CTLA-4 actually regulate immunity. For example, the consequences of TCR signaling in the absence of CD28 are still not clear with anergy, death, and ignorance all viable possibilities. In addition, our current lack of understanding concerning the specificity of the T cells that cause disease in the absence of CTLA-4 is a barrier to understanding how this molecule prevents autoimmunity. We suggest that many of these uncertainties are related to a past reliance on unphysiologic in vitro activation stimuli or monoclonal T cells with fixed affinity for one peptide:MHC (pMHC) ligand. In contrast, we will use a new method based on pMHCII tetramers and magnetic bead enrichment to study CD28 and CTLA-4 effects on polyclonal CD4+ T cells with a normal range of affinities for a pMHCII. In the first aim, we will test the idea that CD28 signals in two distinct modes, one relying on TCR co-clustering and a YMNM motif in the cytoplasmic tail and another involving a PYAP motif that is independent of TCR co-clustering. We hypothesize that the co-clustering mode is critical for pMHCII sensing by clones with low affinity TCRs. These aims will be pursued by measuring clonal expansion, contraction, phenotype conversion, TCR affinity, and memory cell formation by polyclonal pMHCII-specific naive CD4+ T cells lacking one or both of the signaling modes during development and the primary immune response. The second aim is to determine if CTLA-4 constrains the activation of CD4+ T cells expressing TCRs with high affinities for self pMHCII. The hypothesis will be tested in CTLA-4-deficient mice with candidate self pMHCII and by using CD4+ T cell clones from the diseased organs of CTLA-4-deficient mice and an expression cloning system to identify their TCR ligands. This set of experiments has the potential to provide a clear picture of CD28 and CTLA-4 function under physiologically-relevant conditions for T cells with a range of TCR affinities. Narrative: These studies are relevant because they focus on molecules (CD28 and CTLA-4) that regulate the quality of the immune response by T lymphocytes. Several promising therapies based on blockade of these molecules are in use or development for the treatment of arthritis, transplant rejection, and cancer. The plan described in this application is designed to further the understanding of the mechanisms by which CD28 and CTLA-4 control the immune response with the hope of improving the efficacy of the aforementioned treatments and extending them to other T cell-mediated diseases.